This invention relates to a method and device for identifying biomolecular variants. In particular, the invention is concerned with a method for detecting the presence and quantities of biomolecular variants admixed in a sample. Detection of biomolecular variants is accomplished indirectly by assaying the activity of the variants in a sample via reaction product production and/or binding or reaction kinetics with a reactant.
Many diseases are related to variations in the genetically specified sequences of biomolecules. For example, point mutations in a gene can result in the production of a protein variant having an altered structure, and consequently altered activity. This change in protein structure, relative to the native protein molecule, may alter the protein's function as an enzyme, transmitter or antigen, for example, in a manner that directly causes disease. In other cases, the presence of a particular protein variant is a predisposing risk factor or genetic marker for disease. Further, a patient's health or disease is related to the amounts of biomolecules rather than the precise mutation or presence or absence of a biomolecular variant due to gene expression. Thus, it is desirable to have an assay to detect biomolecular variants to diagnose and treat various disease states.
Current molecular biological techniques enable the identification and quantification of biomolecular variants once isolated (for example, by sequencing genes encoding or the amino acids comprising a protein). However, isolating biomolecular variants biomolecules is often difficult. First, biomolecules and their variants are admixed with many different biomolecule populations in body fluids and tissue extracts. Second, the physical properties by which biomolecules are currently isolated (molecular weight, size and charge) are often unchanged or only subtly changed in the mutant variants.
One common laboratory strategy for separating and identifying individual biomolecules from mixtures of large numbers of different biomolecules is to utilize subtle differences in the physical chemistry of those biomolecules to separate them spatially on a surface. For example, chromatographic techniques are used to separate biomolecules based on their molecular weight and diffusibility. This technique can be combined with an orthogonal gradient based on the tendency of the biomolecules to migrate under the influence of an electrical field at a rate proportional to their intrinsic charge. Once separated spatially, localized concentrations of specific biomolecules can be identified, for example by staining them non-specifically for the presence of proteins or nucleic acids or specifically using antibody reactions. However, mutant variants of biomolecules often do not differ sufficiently from native variants in molecular weight, diffusibility or intrinsic charge to be physically separable and identified with these methods.
Currently, the most selective detection methodologies used to detect the presence and quantity of biomolecular variants involve molecule specific binding, between enzymes and substrates or between antigens and antibodies, for example. However, even these molecule-specific reactions often cannot distinguish between small biomolecular variations, such as those caused by point genetic mutations. Thus, assay methods which allow biomolecular variants to be distinguished for identification and quantification are desired.
Mutant variants are more likely to be distinguishable from native variants by their function than their physical chemistry. Biomolecular function is proportionate to a molecule's reaction kinetics with other biochemicals, such as metabolic substrates, degradative enzymes and antibodies. Indeed, these altered reaction kinetics are often related to the pathophysiology of the diseases with which biomolecular variants are associated.
Biomolecular variants can be detected and quantified once spacially or functionally isolated. Sensor arrays for electrochemically detecting the presence and/or reaction kinetics of analytes and biomolecules in solution are described, for example, in U.S. Pat. Nos. 5,891,630, 5,942,388 and 5,951,846, 5,981,203, 5,942,388, 5,906,921, 5,858,799, 5,719,033, 5,753,518, 5,643,721, 5,645,709, 5,653,939, 5,683,569, which are incorporated herein by reference and such technologies may be useful in the present invention. Optically scanned arrays for detecting the binding of biomolecules are also well-known, for example, so-called DNA or micro-array chips. However, there appears no method that systematically alters the reaction kinetics between two biomolecular variants so as to detect the presence or quantify variants of interest. Thus, it is desirable to utilize a method and device to create conditions by which biomolecular variants can be distinguished and quantified to create a rich set of data from which it is possible to distinguish the presence of one or more variants and to quantify the absolute and relative concentrations or amounts of such variants in a sample.